Association of muscle-specific kinase MuSK with the acetylcholine receptor in mammalian muscle.

During synaptogenesis at the neuromuscular junction, a neurally released factor, agrin, causes the clustering of acetylcholine receptors (AChRs) in the muscle membrane beneath the nerve terminal. Agrin acts through a specific receptor which is thought to have a receptor tyrosine kinase, MuSK, as one of its components. In agrin-treated muscle cells, both MuSK and the AChR become tyrosine phosphorylated. To determine how the activation of MuSK leads to AChR clustering, we have investigated their interaction in cultured C2 myotubes. Immunoprecipitation experiments showed that MuSK is associated with the AChR and that this association is increased by agrin treatment. Agrin also caused a transient activation of the AChR-associated MuSK, as demonstrated by MuSK phosphorylation. In agrin-treated myotubes, MuSK phosphorylation increased with the same time course as phosphorylation of the beta subunit of the AChR, but declined more quickly. Although both herbimycin and staurosporine blocked agrin-induced AChR phosphorylation, only herbimycin inhibited the phosphorylation of MuSK. These results suggest that although agrin increases the amount of activated MuSK that is associated with the AChR, MuSK is not directly responsible for AChR phosphorylation but acts through other kinases.     

Agrin-induced activation of acetylcholine receptor-bound Src family kinases requires Rapsyn and correlates with acetylcholine receptor clustering

During neuromuscular synaptogenesis, neurally released agrin induces aggregation and tyrosine phosphorylation of acetylcholine receptors (AChRs) by acting through both the receptor tyrosine kinase MuSK (muscle-specific kinase) and the AChR-associated protein, rapsyn. To elucidate this signaling mechanism, we examined tyrosine phosphorylation of AChR-associated proteins, particularly addressing whether agrin activates Src family kinases bound to the AChR. In C2 myotubes, agrin induced tyrosine phosphorylation of these kinases, of AChR-bound MuSK, and of the AChR beta and delta subunits, as observed in phosphotyrosine immunoblotting experiments. Kinase assays revealed that the activity of AChR-associated Src kinases was increased by agrin, whereas phosphorylation of the total cellular kinase pool was unaffected. In both rapsyn-deficient myotubes and staurosporine-treated C2 myotubes, where AChRs are not clustered, agrin activated MuSK but did not cause either Src family or AChR phosphorylation. In S27 mutant myotubes, which fail to aggregate AChRs, no agrin-induced phosphorylation of AChR-bound Src kinases, MuSK, or AChRs was observed. These results demonstrate first that agrin leads to phosphorylation and activation of AChR-associated Src-related kinases, which requires rapsyn, occurs downstream of MuSK, and causes AChR phosphorylation. Second, this activation intimately correlates with AChR clustering, suggesting that these kinases may play a role in agrin-induced AChR aggregation by forming an AChR-bound signaling cascade.     

Lithium inhibits a late step in agrin-induced AChR aggregation

Agrin activates an intracellular signaling pathway to induce the formation of postsynaptic specializations on muscle fibers. In myotubes in culture, this pathway has been shown to include autophosphorylation of the muscle-specific kinase MuSK, activation of Src-family kinases, tyrosine phosphorylation of the acetylcholine receptor (AChR) beta subunit, a decrease in receptor detergent extractability, and the accumulation of AChRs into high-density aggregates. Here we report that treating chick myotubes with lithium prevented any detectable agrin-induced change in AChR distribution without affecting the number of AChRs or the agrin-induced change in AChR tyrosine phosphorylation and detergent extractability. Lithium treatment also increased the rate at which AChR aggregates disappeared when agrin was removed. The effects of lithium developed slowly over the course of approximately 12 h. Thus, sensitivity to lithium identifies a late step in the agrin signaling pathway, after agrin-induced MuSK and AChR phosphorylation, that is necessary for the recruitment of AChRs into visible aggregates.     

L-type calcium channels mediate acetylcholine receptor aggregation on cultured muscle

Agrin activation of muscle specific kinase (MuSK) initiates postsynaptic development on skeletal muscle that includes the aggregation of acetylcholine receptors (AChRs; Glass et al. [1996]: Cell 85: 513-523; Gautam et al. [1996]: Cell 85: 525-535). Although the agrin/MuSK signaling pathway remains largely unknown, changes in intracellular calcium levels are required for agrin-induced AChR aggregation (Megeath and Fallon [1998]: J Neurosci 18: 672-678). Here, we show that L-type calcium channels (L-CaChs) are required for full agrin-induced aggregation of AChRs and sufficient to induce agrin-independent AChR aggregation. Blockade of L-CaChs in muscle cultures inhibited agrin-induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Activation of L-CaChs in the absence of agrin induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Agrin responsiveness was significantly reduced in primary muscle cultures from the muscular dysgenesis mouse, a natural mutant, which does not express the L-CaCh. Our results establish a novel role for L-CaChs as important sources of the intracellular calcium necessary for the aggregation of AChRs.     

Dual role for calcium in agrin signaling and acetylcholine receptor clustering.

Agrin is a motoneuron-derived factor that initiates neuromuscular synapse formation; however, the signaling pathway underlying postsynaptic differentiation is not yet understood. We have investigated the role of calcium in agrin signaling through the MuSK receptor tyrosine kinase and in the intracellular signaling cascade that leads to AChR phosphorylation and clustering. We find that agrin- and neuramindase-induced MuSK activation in cultured myotubes is completely blocked by removal of extracellular calcium, but only slightly reduced by clamping of intracellular calcium transients with BAPTA. Following agrin's activation of MuSK, we find that the downstream tyrosine phosphorylation of the AChR beta-subunit was inhibited by BAPTA but not by a slower acting chelator, EGTA. Similarly, agrin-induced clustering of the AChR was blocked by BAPTA but not EGTA. These findings indicate that extracellular calcium is required for the formation of a MuSK signaling complex, and that intracellular calcium regulates phosphorylation and clustering of the AChR in the postsynaptic membrane.     

Mechanism of agrin-induced acetylcholine receptor aggregation.

Agrin induces the formation of specializations on chick myotubes in culture at which several components of the postsynaptic apparatus accumulate, including acetylcholine receptors (AChRs). Agrin also induces AChR phosphorylation. Several lines of evidence suggest that agrin-induced phosphorylation of tyrosine residues in the beta subunit of the AChR is an early step in receptor aggregation: agrin-induced phosphorylation and aggregation have the same dose dependence; treatments that prevent aggregation block phosphorylation; phosphorylation begins before any detectable change in receptor distribution, reaches a maximum hours before aggregation is complete, and declines slowly together with the disappearance of aggregates after agrin is withdrawn; agrin slows the rate at which receptors are solubilized from intact myotubes by detergent extraction; and the change in receptor extractability parallels the change in phosphorylation. A model for agrin-induced AChR aggregation is presented in which phosphorylation of AChRs by an agrin-activated protein tyrosine kinase causes receptors to become attached to the cytoskeleton, which reduces their mobility and detergent extractability, and leads to the accumulation of receptors in the vicinity of the activated kinase, forming an aggregate.     

Nicotine decreases agrin signaling and acetylcholine receptor clustering in C2C12 myotube culture

The clustering of acetylcholine receptors (AChRs) in skeletal muscle fibers is a critical event in neuromuscular synaptogenesis. AChRs in concert with other molecules form postsynaptic scaffolds in response to agrin released from motor neurons as motor neurons near skeletal muscle fibers in development. Agrin drives an intracellular signaling pathway that precedes AChR clustering and includes the tyrosine phosphorylation of AChRs. In C2C12 myotube culture, agrin application stimulates the agrin signaling pathway and AChR clustering. Previous studies have determined that the frequency of spontaneous AChR clustering is decreased and AChRs are partially inactivated when bound by the acetylcholine agonist nicotine. We hypothesized that nicotine interferes with AChR clustering and consequent postsynaptic scaffold formation. In the present study, C2C12 myoblasts were cultured with growth medium to stimulate proliferation and then differentiation medium to stimulate fusion into myotubes. They were bathed in a physiologically relevant concentration of nicotine and then subject to agrin treatment after myotube formation. Our results demonstrate that nicotine decreases agrin-induced tyrosine phosphorylation of AChRs and decreases the frequency of spontaneous as well as agrin-induced AChR clustering. We conclude that nicotine interferes with postsynaptic scaffold formation by preventing the tyrosine phosphorylation of AChRs, an agrin signaling event that precedes AChR clustering.     

Phosphoinositide 3-kinase acts through RAC and Cdc42 during agrin-induced acetylcholine receptor clustering

The formation of the neuromuscular junction (NMJ) is regulated by the nerve-derived heparan sulfate proteoglycan agrin and the muscle-specific kinase MuSK. Agrin induces a signal transduction pathway via MuSK, which promotes the reorganization of the postsynaptic muscle membrane. Activation of MuSK leads to the phosphorylation and redistribution of acetylcholine receptors (AChRs) and other postsynaptic proteins to synaptic sites. The accumulation of high densities of AChRs at postsynaptic regions represents a hallmark of NMJ formation and is required for proper NMJ function. Here we show that phosphoinositide 3-kinase (PI3-K) represents a component of the agrin/MuSK signaling pathway. Muscle cells treated with specific PI3-K inhibitors are unable to form full-size AChR clusters in response to agrin and AChR phosphorylation is reduced. Moreover, agrin-induced activation of Rac and Cdc42 is impaired in the presence of PI3-K inhibitors. PI3-K is localized to the postsynaptic muscle membrane consistent with a role during agrin/MuSK signaling. These results put PI3-K downstream of MuSK as regulator of AChR phosphorylation and clustering. Its role during agrin-stimulated Rac and Cdc42 activation suggests a critical function during cytoskeletal reorganizations, which lead to the redistribution of actin-anchored AChRs.     

Synaptic differentiation is defective in mice lacking acetylcholine receptor beta-subunit tyrosine phosphorylation

Agrin activates MuSK, a receptor tyrosine kinase expressed in skeletal muscle, leading to tyrosine phosphorylation of the acetylcholine receptor (AChR) beta-subunit and clustering of AChRs. The importance of AChR beta-subunit tyrosine phosphorylation in clustering AChRs and regulating synaptic differentiation is poorly understood. We generated mice with targeted mutations in the three intracellular tyrosines of the AChR beta-subunit (AChR-beta(3F/3F)). Mice lacking AChR beta-subunit tyrosine phosphorylation thrive postnatally and have no overt behavioral defects, indicating that AChR beta-subunit tyrosine phosphorylation is not essential for the formation of neuromuscular synapses. Nonetheless, the size of synapses and the density of synaptic AChRs are reduced in AChR- beta(3F/3F) mutant mice. Moreover, synapses are structurally simplified and the organization of postjunctional folds is aberrant in mice lacking tyrosine phosphorylation of the AChR beta-subunit. Furthermore, mutant AChRs cluster poorly in response to agrin and are readily extracted from the cell surface of cultured myotubes by non-ionic detergent. These data indicate that tyrosine phosphorylation of the AChR beta-subunit has an important role in organizing AChRs and regulating synaptic differentiation.     

Modulation of Agrin-induced Acetylcholine Receptor Clustering by Extracellular Signal-regulated Kinases 1 and 2 in Cultured Myotubes

Agrin released by motoneurons induces and/or maintains acetylcholine receptor (AChR) clustering and other aspects of postsynaptic differentiation at the vertebrate neuromuscular junction. Agrin acts by binding and activating a receptor complex containing LDL receptor protein 4 (Lrp4) and muscle-specific kinase (MuSK). Two critical downstream components of this signaling cascade, Dox-7 and rapsyn, have been identified. However, additional intracellular essential elements remain unknown. Prior observations by others and us suggested antagonistic interactions between agrin and neuregulin-1 (Nrg-1) signaling in cultured myotubes and developing muscle fibers in vivo. A hallmark of Nrg-1 signaling in skeletal muscle cells is the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2 are also activated in most cells by phorbol 12-myristate 13-acetate, a classical inhibitor of agrin-induced AChR clustering in myotubes. Here, it was investigated whether agrin activates ERK1/2 directly and whether such activation modulates agrin-induced AChR clustering. Agrin induced a rapid but transient activation of ERK1/2 in myotubes that was Lrp4/MuSK-dependent. However, blocking this ERK1/2 activation did not prevent but potentiated AChR clustering induced by agrin. ERK1/2 activation was dispensable for Nrg-1-mediated inhibition of the AChR clustering activity of agrin, but was indispensable for such activity by phorbol 12-myristate 13-acetate. Together, these results suggest agrin-induced activation of ERK1/2 is a negative modulator of agrin signaling in skeletal muscle cells.     

Mechanism of agrin-induced acetylcholine receptor aggregation

Agrin induces the formation of specializations on chick myotubes in culture at which several components of the postsynaptic apparatus accumulate, including acetylcholine receptors (AChRs). Agrin also induces AChR phosphorylation. Several lines of evidence suggest that agrininduced phosphorylation of tyrosine residues in the β subunit of the AChR is an early step in receptor aggregation: agrin-induced phosphorylation and aggregation have the same dose dependence; treatments that prevent aggregation block phosphorylation; phosphorylation begins before any detectable change in receptor distribution, reaches a maximum hours before aggregation is complete, and declines slowly together with the disappearance of aggregates after agrin is withdrawn; agrin slows the rate at which receptors are solubilized from intact myotubes by detergent extraction; and the change in receptor extractability parallels the change in phosphorylation. A model for agrin-induced AChR aggregation is presented in which phosphorylation of AChRs by an agrin-activated protein tyrosine kinase causes receptors to become attached to the cytoskeleton, which reduces their mobility and detergent extractability, and leads to the accumulation of receptors in the vicinity of the activated kinase, forming an aggregate. © 1992 John Wiley & Sons, Inc.     

Laminin-1 redistributes postsynaptic proteins and requires rapsyn,tyrosine phosphorylation,and Src and Fyn to stably cluster acetylcholine receptors

Clustering of acetylcholine receptors (AChRs) is a critical step in neuromuscular synaptogenesis, and is induced by agrin and laminin which are thought to act through different signaling mechanisms. We addressed whether laminin redistributes postsynaptic proteins and requires key elements of the agrin signaling pathway to cause AChR aggregation. In myotubes, laminin-1 rearranged dystroglycans and syntrophins into a laminin-like network, whereas inducing AChR-containing clusters of dystrobrevin, utrophin, and, to a marginal degree, MuSK. Laminin-1 also caused extensive coclustering of rapsyn and phosphotyrosine with AChRs, but none of these clusters were observed in rapsyn -/- myotubes. In parallel with clustering, laminin-1 induced tyrosine phosphorylation of AChR beta and delta subunits. Staurosporine and herbimycin, inhibitors of tyrosine kinases, prevented laminin-induced AChR phosphorylation and AChR and phosphotyrosine clustering, and caused rapid dispersal of clusters previously induced by laminin-1. Finally, laminin-1 caused normal aggregation of AChRs and phosphotyrosine in myotubes lacking both Src and Fyn kinases, but these clusters dispersed rapidly after laminin withdrawal. Thus, laminin-1 redistributes postsynaptic proteins and, like agrin, requires tyrosine kinases for AChR phosphorylation and clustering, and rapsyn for AChR cluster formation, whereas cluster stabilization depends on Src and Fyn. Therefore, the laminin and agrin signaling pathways overlap intracellularly, which may be important for neuromuscular synapse formation.     

Lithium inhibits a late step in agrin‐induced AChR aggregation

Agrin activates an intracellular signaling pathway to induce the formation of postsynaptic specializations on muscle fibers. In myotubes in culture, this pathway has been shown to include autophosphorylation of the muscle‐specific kinase MuSK, activation of Src‐family kinases, tyrosine phosphorylation of the acetylcholine receptor (AChR) β subunit, a decrease in receptor detergent extractability, and the accumulation of AChRs into high‐density aggregates. Here we report that treating chick myotubes with lithium prevented any detectable agrin‐induced change in AChR distribution without affecting the number of AChRs or the agrin‐induced change in AChR tyrosine phosphorylation and detergent extractability. Lithium treatment also increased the rate at which AChR aggregates disappeared when agrin was removed. The effects of lithium developed slowly over the course of approximately 12 h. Thus, sensitivity to lithium identifies a late step in the agrin signaling pathway, after agrin‐induced MuSK and AChR phosphorylation, that is necessary for the recruitment of AChRs into visible aggregates. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 346–357, 2003     

Regulation of the interaction of nicotinic acetylcholine receptors with the cytoskeleton by agrin-activated protein tyrosine kinase

Agrin induces the accumulation of nicotinic acetylcholine receptors (AChRs) in the myofiber membrane at synaptic sites in vertebrate skeletal muscle and causes an increase in tyrosine phosphorylation of the AChR beta subunit. To examine further the mechanism of agrin- induced AChR phosphorylation and the relationship between changes in protein phosphorylation and AChR aggregation, the effect of the protein tyrosine phosphatase inhibitor sodium pervanadate was tested on chick myotubes in culture. Pervanadate caused an increase in the phosphotyrosine content of a variety of proteins, including the AChR. Pervanadate also prevented agrin-induced AChR aggregation and slowed the rate at which AChRs were extracted from intact myotubes by mild detergent treatment. The rate at which phosphorylation of the AChR beta subunit and receptor detergent extractability changed following pervanadate-induced phosphatase inhibition was increased by agrin, indicating that agrin activates a protein tyrosine kinase rather than inhibiting a protein tyrosine phosphatase. The present results, taken together with previous findings on the inhibition of agrin-induced AChR aggregation by protein kinase inhibitors, demonstrate that protein tyrosine phosphorylation regulates the formation and stability of AChR aggregates, apparently by strengthening the interaction between AChRs and the cytoskelton.     

Agrin induces phosphorylation of the nicotinic acetylcholine receptor.

Agrin causes acetylcholine receptors (AChRs) on chick myotubes in culture to aggregate, forming specializations that resemble the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction. Here we report that treating chick myotubes with agrin caused an increase in phosphorylation of the AChR beta, gamma, and delta subunits. H-7, a potent inhibitor of several protein serine kinases, blocked agrin-induced phosphorylation of the gamma and delta subunits, but did not prevent either agrin-induced AChR aggregation or phosphorylation of the beta subunit. Experiments with anti-phosphotyrosine antibodies demonstrated that agrin caused an increase in tyrosine phosphorylation of the beta subunit that began within 30 min of adding agrin to the myotube cultures, reached a plateau by 3 hr, and was blocked by treatments known to block agrin-induced AChR aggregation. Anti-phosphotyrosine antibodies labeled agrin-induced specializations as they do the postsynaptic apparatus. These results suggest that agrin-induced tyrosine phosphorylation of the beta subunit may play a role in regulating AChR distribution.     

Regulation of ACh receptor clustering by the tyrosine phosphatase Shp2

At the vertebrate neuromuscular junction (NMJ), postsynaptic aggregation of muscle acetylcholine receptors (AChRs) depends on the activation of MuSK, a muscle-specific tyrosine kinase that is stimulated by neural agrin and regulated by muscle-intrinsic tyrosine kinases and phosphatases. We recently reported that Shp2, a tyrosine phosphatase containing src homology two domains, suppressed MuSK-dependent AChR clustering in cultured myotubes, but how this effect of Shp2 is controlled has remained unclear. In this study, biochemical assays showed that agrin-treatment of C2 mouse myotubes enhanced the tyrosine phosphorylation of signal regulatory protein alpha1 (SIRPalpha1), a known activator of Shp2, and promoted SIRPalpha1's interaction with Shp2. Moreover, in situ experiments revealed that treatment of myotubes with the Shp2-selective inhibitor NSC-87877 increased spontaneous and agrin-induced AChR clustering, and that AChR clustering was also enhanced in myotubes ectopically expressing inactive (dominant-negative) Shp2; in contrast, AChR clustering was reduced in myotubes expressing constitutively active Shp2. Significantly, expression of truncated (nonShp2-binding) and full-length (Shp2-binding) forms of SIRPalpha1 in myotubes also increased and decreased AChR clustering, respectively, and coexpression of truncated SIRPalpha1 with active Shp2 and full-length SIRPalpha1 with inactive Shp2 reversed the actions of the exogenous Shp2 proteins on AChR clustering. These results suggest that SIRPalpha1 is a novel downstream target of MuSK that activates Shp2, which, in turn, suppresses AChR clustering. We propose that an inhibitory loop involving both tyrosine kinases and phosphatases sets the level of agrin/MuSK signaling and constrains it spatially to help generate high-density AChR clusters selectively at NMJs.     

Dual role for calcium in agrin signaling and acetylcholine receptor clustering

Agrin is a motoneuron‐derived factor that initiates neuromuscular synapse formation; however, the signaling pathway underlying postsynaptic differentiation is not yet understood. We have investigated the role of calcium in agrin signaling through the MuSK receptor tyrosine kinase and in the intracellular signaling cascade that leads to AChR phosphorylation and clustering. We find that agrin‐ and neuramindase‐induced MuSK activation in cultured myotubes is completely blocked by removal of extracellular calcium, but only slightly reduced by clamping of intracellular calcium transients with BAPTA. Following agrin's activation of MuSK, we find that the downstream tyrosine phosphorylation of the AChR β‐subunit was inhibited by BAPTA but not by a slower acting chelator, EGTA. Similarly, agrin‐induced clustering of the AChR was blocked by BAPTA but not EGTA. These findings indicate that extracellular calcium is required for the formation of a MuSK signaling complex, and that intracellular calcium regulates phosphorylation and clustering of the AChR in the postsynaptic membrane. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 69–79, 2002     

Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation

Agrin, a protein that mediates nerve-induced acetylcholine receptor (AChR) aggregation at developing neuromuscular junctions, has been shown to cause an increase in phosphorylation of the beta, gamma, and delta subunits of AChRs in cultured myotubes. As a step toward understanding the mechanism of agrin-induced AChR aggregation, we examined the effects of inhibitors of protein kinases on AChR aggregation and phosphorylation in chick myotubes in culture. Staurosporine, an antagonist of both protein serine and tyrosine kinases, blocked agrin-induced AChR aggregation in a dose-dependent manner; 50% inhibition occurred at approximately 2 nM. The extent of inhibition was independent of agrin concentration, suggesting an effect downstream of the interaction of agrin with its receptor. Staurosporine blocked agrin-induced phosphorylation of the AChR beta subunit, which occurs at least in part on tyrosine residues, but did not reduce phosphorylation of the gamma and delta subunits, which occurs on serine/threonine residues. Staurosporine also prevented the agrin- induced decrease in the rate at which AChRs are extracted from intact myotubes by mild detergents. H-7, an antagonist of protein serine kinases, inhibited agrin-induced phosphorylation of the gamma and delta subunits but did not block agrin-induced phosphorylation of the AChR beta subunit, AChR aggregation, or the decrease in AChR extractability. The results provide support for the hypothesis that tyrosine phosphorylation of the beta subunit plays a role in agrin-induced AChR aggregation.     

Conjugation of LG domains of agrins and perlecan to polymerizing laminin-2 promotes acetylcholine receptor clustering

Neuromuscular junction (NMJ) assembly is characterized by the clustering and neuronal alignment of acetylcholine receptors (AChRs). In this study we have addressed post-synaptic contributions to assembly that may arise from the NMJ basement membrane with cultured myotubes. We show that the cell surface-binding LG domains of non-neural (muscle) agrin and perlecan promote AChR clustering in the presence of laminin-2. This type of AChR clustering occurs with a several hour lag, requires muscle-specific kinase (MuSK), and is accompanied by tyrosine phosphorylation of MuSK and betaAChR. It also requires conjugation of the agrin or perlecan to laminin together with laminin polymerization. Furthermore, AChR clustering can be mimicked with antibody binding to non-neural agrin, supporting a mechanism of ligand aggregation. Neural agrin, in addition to its unique ability to cluster AChRs through its B/z sequence insert, also exhibits laminin-dependent AChR clustering, the latter enhancing and stabilizing its activity. Finally, we show that type IV collagen, which lacks clustering activity on its own, stabilizes laminin-dependent AChR clusters. These findings provide evidence for cooperative and partially redundant MuSK-dependent functions of basement membrane in AChR assembly that can enhance neural agrin activity yet operate in its absence. Such interactions may contribute to the assembly of aneural AChR clusters that precede neural agrin release as well as affect later NMJ development.     

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.